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'Genotyping Microarray for CSNB-Associated Genes' Zeitz, C; Labs, S; Lorenz, B; Forster, U; Üksti, J; Kroes, H Y; De Baere, E; Leroy, B P; Cremers, F P M; Wittmer, M; van Genderen, M M; Sahel, J A; Audo, I; Poloschek, C M; Mohand-Said, S; Fleischhauer, J C; Hüffmeier, U; Moskova-Doumanova, V; Levin, A V; Hamel, C P; Leifert, D; Munier, F L; Schorderet, D F; Zrenner, E; Friedburg, C; Wissinger, B; Kohl, S; Berger, W (2009). Genotyping Microarray for CSNB-Associated Genes. Investigative Ophthalmology and Visual Science, 12(50):5919-5926. University of Zurich Postprint available at: Zurich Open Repository and Archive http://www.zora.uzh.ch Posted at the Zurich Open Repository and Archive, University of Zurich. http://www.zora.uzh.ch Winterthurerstr. 190 Originally published at: CH-8057 Zurich Investigative Ophthalmology and Visual Science 2009, 12(50):5919-5926. http://www.zora.uzh.ch Year: 2009 Genotyping Microarray for CSNB-Associated Genes Zeitz, C; Labs, S; Lorenz, B; Forster, U; Üksti, J; Kroes, H Y; De Baere, E; Leroy, B P; Cremers, F P M; Wittmer, M; van Genderen, M M; Sahel, J A; Audo, I; Poloschek, C M; Mohand-Said, S; Fleischhauer, J C; Hüffmeier, U; Moskova-Doumanova, V; Levin, A V; Hamel, C P; Leifert, D; Munier, F L; Schorderet, D F; Zrenner, E; Friedburg, C; Wissinger, B; Kohl, S; Berger, W Zeitz, C; Labs, S; Lorenz, B; Forster, U; Üksti, J; Kroes, H Y; De Baere, E; Leroy, B P; Cremers, F P M; Wittmer, M; van Genderen, M M; Sahel, J A; Audo, I; Poloschek, C M; Mohand-Said, S; Fleischhauer, J C; Hüffmeier, U; Moskova-Doumanova, V; Levin, A V; Hamel, C P; Leifert, D; Munier, F L; Schorderet, D F; Zrenner, E; Friedburg, C; Wissinger, B; Kohl, S; Berger, W (2009). Genotyping Microarray for CSNB-Associated Genes. Investigative Ophthalmology and Visual Science, 12(50):5919-5926. Postprint available at: http://www.zora.uzh.ch Posted at the Zurich Open Repository and Archive, University of Zurich. http://www.zora.uzh.ch Originally published at: Investigative Ophthalmology and Visual Science 2009, 12(50):5919-5926. Genotyping Microarray for CSNB-Associated Genes Abstract PURPOSE. Congenital stationary night blindness (CSNB) is a clinically and genetically heterogeneous retinal disease. Although electroretinographic (ERG) measurements can discriminate clinical subgroups, the identification of the underlying genetic defects has been complicated for CSNB because ofgenetic heterogeneity, the uncertainty about the mode of inheritance, and time-consuming and costly mutation scanning and direct sequencing approaches. METHODS. To overcome these challenges and to generate a time- and cost-efficient mutation screening tool, the authors developed a CSNB genotyping microarray with arrayed primer extension (APEX) technology. To cover as many mutations as possible, a comprehensive literature search was performed, and DNA samples from a cohort of patients with CSNB were first sequenced directly in known CSNB genes. Subsequently, oligonucleotides were designed representing 126 sequence variations in RHO, CABP4, CACNA1F, CACNA2D4, GNAT1,GRM6, NYX, PDE6B, and SAG and spotted on the chip. RESULTS. Direct sequencing of genes known to be associated with CSNB in the study cohort revealed 21 mutations (12 novel and 9 previously reported). The resultant microarray containing oligonucleotides, which allow to detect 126 known and novel mutations, was 100% effective in determining the expected sequence changes in all known samples assessed. In addition, investigation of 34 patients with CSNB who were previously not genotyped revealed sequence variants in 18%, of which 15% are thought to be disease-causing mutations. CONCLUSIONS. This relatively inexpensive first-pass genetic testing device for patients with a diagnosis of CSNB will improve molecular diagnostics and genetic counseling of patients and their families and gives the opportunity to analyze whether, for example, more progressive disorders such as cone or cone-rod dystrophies underlie the same gene defects. Genotyping Microarray for CSNB-Associated Genes Christina Zeitz,1,2,3 Stephan Labs,1 Birgit Lorenz,4 Ursula Forster,1 Janne U¨ksti,5 Hester Y. Kroes,6 Elfride De Baere,7 Bart P. Leroy,7,8 Frans P. M. Cremers,9,10 Mariana Wittmer,1 Maria M. van Genderen,11 Jose´-Alain Sahel,2,3,12 Isabelle Audo,2,3,12 Charlotte M. Poloschek,13 Saddek Mohand-Saïd,12 Johannes C. Fleischhauer,14 Ulrike Hu¨ffmeier,15 Veselina Moskova-Doumanova,2,3 Alex V. Levin,16 Christian P. Hamel,17 Dorothee Leifert,18 Francis L. Munier,19 Daniel F. Schorderet,20 Eberhart Zrenner,21 Christoph Friedburg,4 Bernd Wissinger,22 Susanne Kohl,22 and Wolfgang Berger1 PURPOSE. Congenital stationary night blindness (CSNB) is a though electroretinographic (ERG) measurements can discrim- clinically and genetically heterogeneous retinal disease. Al- inate clinical subgroups, the identification of the underlying genetic defects has been complicated for CSNB because of genetic heterogeneity, the uncertainty about the mode of in- 1 heritance, and time-consuming and costly mutation scanning From the Division of Medical Molecular Genetics and Gene Diag- and direct sequencing approaches. nostics, Institute of Medical Genetics, University of Zurich, Zurich, Swit- zerland; 2INSERM (Institut National de la Sante´ et de la Recherche Me´di- METHODS. To overcome these challenges and to generate a cale), UMR_S968, and the 3Department of Genetics, Institut de la Vision, time- and cost-efficient mutation screening tool, the authors Universite´ Pierre et Marie Curie (UPMC), Universite´ Paris 06, Paris, France; developed a CSNB genotyping microarray with arrayed primer the 4Department of Ophthalmology, Justus-Liebig-University Giessen, Uni- extension (APEX) technology. To cover as many mutations as versita¨tsklinikum Giessen and Marburg, Giessen Campus, Giessen, Ger- 5 6 possible, a comprehensive literature search was performed, many; Asper Biotech, Tartu, Estonia; the Department of Medical Genet- and DNA samples from a cohort of patients with CSNB were ics, University Medical Center Utrecht, Utrecht, The Netherlands; the 7Center for Medical Genetics and 8Department of Ophthalmology, Ghent first sequenced directly in known CSNB genes. Subsequently, University Hospital, Ghent, Belgium; the 9Department of Human Genetics, oligonucleotides were designed representing 126 sequence Radboud University Nijmegen Medical Centre, Nijmegen, The Nether- variations in RHO, CABP4, CACNA1F, CACNA2D4, GNAT1, lands; the 10Nijmegen Centre for Molecular Life Sciences, Radboud Uni- GRM6, NYX, PDE6B, and SAG and spotted on the chip. versity Nijmegen, Nijmegen, The Netherlands; the 11Institute for the 12 RESULTS. Direct sequencing of genes known to be associated Visually Impaired, Zeist, The Netherlands; INSERM CIC 503, Centre with CSNB in the study cohort revealed 21 mutations (12 novel Hospitalier National d’Ophtalmologie des Quinze-Vingts, Paris, France; the 13Department of Ophthalmology, University of Freiburg, Freiburg, Ger- and 9 previously reported). The resultant microarray contain- many; the 14Department of Ophthalmology, University Hospital Bern, ing oligonucleotides, which allow to detect 126 known and Bern, Switzerland; the 15Institute for Human Genetics, University Erlan- novel mutations, was 100% effective in determining the ex- gen, Germany; the 16Pediatric Ophthalmology and Ocular Genetics, Wills pected sequence changes in all known samples assessed. In Eye Institute, Philadelphia, Pennsylvania; 17INSERM U 583, Physiopatholo- addition, investigation of 34 patients with CSNB who were gie et The´rapie des De´ficits Sensoriels et Moteurs, Institut des Neuro- previously not genotyped revealed sequence variants in 18%, sciences de Montpellier, Hoˆpital Saint-Eloi, Montpellier, France; the 18De- of which 15% are thought to be disease-causing mutations. partment of Ophthalmology, University Hospital Basel, Basel, Switzerland; the 19Unit of Oculogenetics, Jules Gonin Eye Hospital, Lausanne, Switzer- CONCLUSIONS. This relatively inexpensive first-pass genetic test- land; the 20Institut de Recherche en Ophtalmologie (IRO), Ecole Polytech- ing device for patients with a diagnosis of CSNB will improve nique Fe´de´rale de Lausanne, University of Lausanne, Sion, Switzerland; molecular diagnostics and genetic counseling of patients and 21The University Eye Clinic and the 22Molecular Genetics Laboratory, their families and gives the opportunity to analyze whether, for Institute for Ophthalmic Research, Centre for Ophthalmology, University example, more progressive disorders such as cone or cone–rod Clinics Tu¨bingen, Tu¨bingen, Germany. dystrophies underlie the same gene defects. (Invest Ophthal- Supported by Forschungeskredit, University of Zurich (CZ), Foun- mol Vis Sci. 2009;50:5919–5926) DOI:10.1167/iovs.09-3548 dation Voir et Entendre (CZ), BQR, UPMC, Universite´ Paris 06 (CZ), DFG Grant ZR1/17-2/KFO 134 (EZ, BW, SK), Swiss National Science Foundation Grant 32-111948/1 (FLM), the Research Foundation ongenital stationary night blindness (CSNB) is a clinically Flanders Grant G.0043.06N (BPL, EDB), and a grant from The Founda- Cand genetically heterogeneous retinal disease. It can be tion Fighting Blindness (IA, SM-S, J-AS). associated with deficiency of vision under dim light conditions, Submitted for publication February 10, 2009; revised April 29 and nystagmus, refractive error, or retinal changes. Electroretinog- June 4, 2009; accepted August 26, 2009. Disclosure: C. Zeitz, None; S. Labs, None; B. Lorenz, None; U. raphy is helpful in confirming and subclassifying the disorder. Forster, None; J. U¨ ksti, None; H.Y. Kroes,
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